Hi Bei, For the extracellular protein I worked on in graduate school, I typically purified it from 4 L preps in LB media. The standard protocol was to do a crude low cut with ammonium sulfate cut followed by precipitation of the protein with a high cut. The pellet was then resuspended, dialyzed and loaded on an S-sepharose column. I experimented with taking the media (after spinning out the cells) and diluting 1:1 with a low ionic strength buffer and loading directly onto the S-sepharose column. This worked, but the loading time for 8 L was so long it was not worth it. Another option might have been to use bulk media to bind the protein in a batch step. I never tested this. A final option that we explored was Expanded-Bed Adsorption Chromatography. This would allow us to get rid of the initial centrifugation step to remove the cells from the media and would allow fast loading with a high speed pump. We priced out the media, column and pump from Pharmacia at the time, but never ended up purchasing the system. The technology looked promising and should have worked well for our system, but we decided that we did not need to do too many more preps for the project and just used the standard protocol. Regards, Mitch
-----Original Message----- From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of joybeiyang Sent: Tuesday, April 12, 2011 2:14 PM To: [email protected] Subject: [ccp4bb] methods to capture proteins from cell culture medium Dear all, My protein of interest was expressed as secreted protein, so I have to collect the medium and change the buffer with sortorius Jet before I load the sample onto a IMAC, the buffer change step in my current protocol can last for 12hrs (I have to concentrate 4L to 200ml, then dilute it with lysis buffer and concentrate it again, then dilute and concentrate repeatedly) and is really boring and troublesome, besides I always observe protein loss during this step and the detergent in the medium usually concentrate as well in this step which would interfere with subsequent purification process. I am wondering if there are more convenient ways to capture the target protein from medium? How about the following: 1. directly load the medium onto a ion exchange column? 2. Amonium sulfate precipitation? 3. anyother thoughts? Thank you very much in advance! Best, Bei 2011-04-12 ________________________________ joybeiyang
